Micropropagation of Anthurium Andreanum Cv. Terra

Full Length Research Paper

Micropropagation of Anthurium andreanum cv. Terra

Farsi M.1, Taghavizadeh Yazdi ME.2* and Qasemiomran V3

1Ferdowsi University of Mashhad, Mashhad, I. R. Iran. 2Department of Plant Sciences, Eram Biotechnology Research Center, Technical and Vocational Training Organization, Mashad, I. R. Iran 3Genetics and agricultural biotechnology institute of Tabarestan, Sari agricultural sciences and natural resources University, Sari, Iran.

Accepted 13 June, 2012

Anthurium anderanum cv. Terra from family Araceae, is a plant with a high commercial value. Conventional propagation of this plant performed via suckers and seeds planting that nowadays missed its application have been abolished. This study aimed to establish producible protocol for indirect in vitro regeneration of the Terra genotype. For this purpose, the effects of 16 different plant growth regulators treatments on callus induction and regeneration of leaf explants were studied. Callus with the highest fresh and dry weight was produced on modified Murashige and Skoog medium containing 0.1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) and 1.5 mg/L 6-benzylaminopurine (BAP). For regeneration stage, induced calli were transferred to modified MS medium without plant growth regulators, and after six weeks, shoots formed. For rooting, regenerated shoot were transferred to MS medium without any plant growth regulators. Regenerated plantlets potted on mixture of coco pit and perlite were succesfully acclimated.

Key words: Anthurium, micropropagation, growth regulators, regeneration.

INTRODUCTION

The Anthurium genus comprises about 1500 tropical regeneration of Anthurium andreanum through species with economically important genera in the family adventitious shoots formation from callus (Pierik et al., of Araceae. Anthurium species are produced for many 1974; Pierik and Steegmans, 1976) and also direct shoot proper goals, including cut-flower, flowering potted plants regeneration from lamina explants were achieved (Martin and landscape plants (Nowbuth et al., 2005). Anthurium et al., 2003). Vargas et al. (2004) established an are traditionally propagated by seeds (Dufour and Guerin, alternative method for regeneration of Anthurium plants. 2003), however, vegetative propagation methods applied They obtained in vitro plants from germinated seeds and to these plants have not shown good results and tissue plantlets from micro-cuttings culture. Joseph et al. (2003) culture techniques appears as an alternative to increase reported the calli induction from leaf explants of the production (Pierik et al., 1974; Chen et al., 1997). Anthurium in modified Murashige and Skoog (MS) Propagation through seeds is not worthwhile because of medium with 0.88 µM 6-benzylaminopurine (BAP) + 0.9 cross-pollination and the progenies are heterozygous. µM 2,4-dichlorophenoxy acetic acid (2,4-D) + 0.46 µM Moreover, it is hampered by the poor germination rate kinetin (Kin). Chen et al. (1997) also regenerated and low viability of the seeds. (Martin et al., 2003). Anthurium plants from root explants. In addition, Yi-xun et Micropropagation of Anthurium has been achieved with al. (2009) induced calli from proto-corm-like of A. various tissues. The tissue culture of Anthurium was first andreanum Hort. Micropropagation by callus induction reported by Pierik et al. (1974). They achieved from leaf explants is a difficult step for in vitro propagation Nevertheless, credible proliferation of callus and subsequent plant regeneration is important for enormous plant propagation. This article describes the *Corresponding author. E-mail: [email protected]. establishment of rapid method for regeneration of

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Table 1. Growth regulator composition of culture media Callus induction used in callus induction (mg/L). Leaf explants were transplanted into dishes supplemented with 30 Treatment 2,4-D BAP g/L sucrose, and different treatment of 2,4-D and BAP. The pH was adjusted to 5.7 before autoclaving. The media differed in 1 0.5 - concentrations of plant growth regulators and light conditions of 2 1 - culture. 3 1.5 - 4 2 - 5 - 0.5 Shoot regeneration 6 - 0.7 For shoot regeneration, calli of leaf explants were transferred to 7 - 1 modify MS with 0.1 mg/L 2, 4-D and 1 mg/L BAP, and kept at 16/8 8 - 1.5 light and darkness photoperiod. 9 - 2 10 - 2.5 11 0.1 0.5 Rooting 12 0.1 1 The regenerated shoots longer than 3 cm with a pair of leaves were 13 0.08 0.5 transferred to the MS medium without plant growth regulators. 14 0.08 1.5 15 0.1 1.5 16 0.16 1.5 Statistical analysis

The results are presented as mean values ± standard errors. All experiments were repeated four times. The data on callus induction and wet and dry weight were subjected to analysis of SPSS ver19, Table 2. The effects of BAP on callus induction. ' with the means separation (p<0.05) by Duncan s multiple range test. The charts were drawn by Excel 2007 (Table 1). BAP (mg/l) Percentage of callus genesis e 0.5 30 0.7 50d RESULTS 1 60c 1.5 80b Callus formation of Anthurium was observed on the leaf 2 80b explants using modified MS medium. The stable of leaf 2.5 90a explants in MS medium was on the average. Callus formation was performed 60 days after culture of Means followed by the same case letters are not significantly different at the 5% probability level by Duncan's new multiple- explants. Table 1 shows the rate of growth regulators range test. used for callus induction from leaves. 2,4-D alone had no effect on callus induction of Anthurium. The effects of BAP on the percentage of callus formation are given in Table 2. Maximum of callus genesis with BAP occurred at Anthurium andreanum cv Terra from callus tissue through a concentration of 2.5 mg/L. The percentage of callus organogenesis. induction with BAP differed significantly (P<0.05). Moreover, the best medium for callus induction from leaf explants was modified MS with 0.1 mg/L 2,4-D +1.5 mg/L MATERIALS AND METHODS BAP. Interplay of 2,4-D and BAP was significant (P<0.05). Figure 2 shows the rate of wet and dry weight Commercial cultivar Terra from A. andreanum was selected. The in the different plant growth regulators treatments. Leaf leaf explants from this variety was used as plant material. The explants cultured on half strength MS were ruined finally. leaves were sterilized for 20 min in 3% hypochlorite sodium and then rinsed three times with sterile water. Sterile leaves were Explants were transferred to 16/8 light and dark sectioned to about 11 cm2. photoperiod to enhance organogenesis in calli. After four weeks, pink organs were formed which showed negative gravitropism, and after two weeks, leaf primordial were Media engendered. For rooting, the regenerated shoot from callus was transferred to MS medium without any plant For the propagation by indirect organogenesis, MS medium (Murashige and Skoog, 1962), half strength MS and modified MS growth regulators. Figure 1 shows the callus induction from leaf explants, organogenesis, regeneration and (half strength of MS medium except of MgSO4 and CaCl2) were used. compatibility of plantlets.

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a b c

d e f

Figure 1. Shows the callus induction from leaf explants, organogenesis, regeneration and compatibility of plantlets.

DISCUSSION choice of explants material (George et al., 2008). In this research, we used leaf explants. On the other hand, Te- Many investigators have reported that the vegetative chato et al. (2006) used leaves, node and internodes of propagation of many Anthurium species is a very difficult three genotypes of Anthurium. They cultured explants on method (Hamidah et al., 1997; Pierik and Steegmans, MS medium, modified MS, woody plant medium (WPM) 1976; Pierik et al., 1974). Micropropagation is an and Nitsch and Nitsch medium (NN) and obtained the alternative method of propagation that is appropriate for highest callus formation from leaf explants in MS and selection of spices. In this research, we achieved a rapid modified MS. Moreover, Nhut et al. (2006) studied the regeneration system of A. andreanum from callus culture. effect of ten different Anthurium genotypes and reported Nutrition, genotypes and growth regulations are the most different responses from their genotype. Our research important cultural factors that a commercial Anthuriums also show that the application of 2,4-D with BAP could be grower can control. Silva et al. (2005) concluded that positive for the propagation of A. andreanum cv. Terra. various physical and biological factors including media Cimen and Ozge (2009) used 1 mg/L IBA and 0.04% play important roles in propagation of A. andreanum. active charcoals to initiate roots. In addition, Joseph et al. Geier (1986) analyzed the influence of NH4NO3 on callus (2003) transferred shoots regenerated from A. and shoot formation from leaf tissues. Leaf explants in andreanum Hort. onto half strength MS medium MS medium had average stability, whereas those in half supplemented with 0.54 µM of naphthaleneacetic acid strength of MS medium showed deficiency of nutrient (NAA) for rooting. The rooting process occurred on material and were ruined finally. The rate of NH4NO3 in medium without plant growth regulators. Plantlets potted MS medium appeared high in callus induction of on mixture of coco pit and perlit were succesfully Anthuriums and must be lower (Joseph et al., 2003; acclimated. Cimen and Ozge, 2009). With lower rate of NH4NO3, the This paper establishes a rapid method for calli induction occurred. Our investigation also showed of micropropagation of A. andreanum cv. Terra. We course that BAP can induce callus, but when used with concluded that in addition to mineral salt of mediums and 2,4-D, had more effect on callus induction. plant growth regulators, age and genotype of explants The success of tissues culture is related to the correct are very important in the success of plant tissue culture

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0.016

weight (g) weight

ry ry

Figure 2. The effects of various hormonal compounds on fresh and dry weights of induced calli. Each bar represents the mean of 20 replications. Bars indicated by similar letters are not significantly different at P = 0.05 (Duncan's new multiple-range test).

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